Synthetic gene encoding human epidermal growth factor 2/neu antigen and uses thereof

ABSTRACT

Synthetic polynucleotides encoding human HER2/neu or a truncated form thereof, are provided, the synthetic polynucleotides being codon-optimized for expression in a human cellular environment. The gene encoding hHER2 is commonly associated with the development of human carcinomas. The present invention provides compositions and methods to elicit or enhance immunity to the protein product expressed by the hHER2 tumor-associated antigen, wherein aberrant hHER2 expression is associated with a carcinoma or its development. This invention specifically provides adenoviral vector and plasmid constructs carrying codon-optimized human HER2 and codon-optimized truncated HER2, and discloses their use in vaccines and pharmaceutical compositions for preventing and treating cancer.

FIELD OF THE INVENTION

The present invention relates generally to the therapy of cancer. Morespecifically, the present invention relates to synthetic polynucleotidesencoding the human tumor associated polypeptide epidermal growth factor2/neu antigen, herein designated hHER2.opt, wherein the polynucleotidesare codon-optimized for expression in a human cellular environment. Thepresent invention also relates to synthetic polynucleotides encoding atruncated form of the HER2/neu antigen, herein designatedhHER2ECDTM.opt, wherein the polynucleotides are codon-optimized forexpression in a human cellular environment. The present inventionfurther relates to recombinant vectors and hosts comprising saidsynthetic polynucleotides. This invention also provides adenoviralvector and plasmid constructs carrying hHER2.opt and to their use invaccines and pharmaceutical compositions for preventing and treatingcancer.

BACKGROUND OF THE INVENTION

Epidermal growth factor 2 is a transmembrane tumor associated antigenencoded by the HER2/neu proto-oncogene (also called c-erbB-2), which isa member of the epidermal growth factor receptor family of cell surfacereceptors. The HER2 gene was originally isolated from a ratneuroglioblastoma (Shih et al., Nature 290: 261-264 (1981)) and latercloned and characterized from human cells (Coussens et al., Science 230:1132-39 (1985); King et al., Science 229: 974-76 (1985)).

HER2/neu is further classified as a member of the HER family of receptortyrosine kinases, which consists of four receptors that participate incell growth and differentiation. The HER receptors contribute tomaintaining normal cell growth by binding growth factor ligands asdimers. Specifically, human HER2 forms heterodimers with other membersof the EGFR family (HER1, HER3 and HER4) (Klapper et al. Adv Cancer Res77: 25-79 (2000)). Following hHER2 dimerization and tyrosineauto-phosphorylation, docking sites for cytoplasmic signaling moleculesare generated and recruitment of second signaling molecules isinitiated. Intracellular signaling cascades, which ultimately result inthe activation of genes important in cell growth, are thus initiated.

Low levels of expression of the HER2/neu transcript and the encoded 185kD protein are normally detected in adult epithelial cells of varioustissues, including the skin and breast, and tissues of thegastrointestinal, reproductive and urinary tracts (Press et al.,Oncogene 5: 953-962 (1990)). Higher levels of HER2/neu expression arealso detected in the corresponding fetal tissues during embryonicdevelopment (Press et al., supra).

Several observations make the HER2 antigen an attractive target foractive specific immunotherapy. First, the HER2/neu gene is commonlyoverexpressed or amplified in various malignancies, such as carcinomasof the breast, ovary, uterus, colon, and prostate, and adenocarcinomasof the lung (reviewed in Disis and Cheever, Adv. Cancer Research 71:343-371 (1997)). Overexpression of HER2/neu correlates with a poorprognosis and a higher relapse rate for cancer patients (Slamon et al.,Science 244: 707-712 (1989)). Amplification of human HER2 leads toenhanced MAP kinase activity and cell proliferation, and contributes tothe aggressive behavior of tumor cells (Ben-Levy et al. Embo J 13(14):3302-11 (1994)). The high expression level of HER2 observed in tumors isin direct contrast with the low levels associated with normal adulttissues.

Additionally, many cancer patients suffering from malignanciesassociated with HER2/neu overexpression have had immune responsesagainst the HER2 protein. Anti-hHER2 cytotoxic T lymphocytes (CTL) havebeen isolated from breast and ovarian cancer patients (loannides et al.Cell Immunol 151(1): 225-34 (1993); Peoples et al. Proc Natl Acad SciUSA 92 (14): 6547-51 (1995)). Several HLA-A2.1-associated hHER2 peptideshave been defined and peptide-specific T cells can be generated in vitro(Fisk et al. Cancer Res 57(1): 8-93 (1997); Yoshino et al. Cancer Res54(13): 3387-90 (1994); Lustgarten et al. Hum Immunol 52(2): 109-18(1997)).

The above findings demonstrate that anti-ErbB-2 immune effectormechanisms are activated in cancer patients and highlight the potentialbenefit of enhancing such immune reactivity. An effective vaccineexploiting the immune response to HER2/neu must both enhance thisimmunity to a level that is protective and/or preventive and overcomeself-tolerance.

Based on the above recitation, HER2/neu has been pursued as a target forthe development of immunological treatments of malignancies. Anti-HER2monoclonal antibodies have been investigated as therapies for breastcancer, with each antibody approach demonstrating various levels ofsuccess (for discussion, see Yarden, Oncology 61(suppl 2): 1-13 (2001)).

Additionally, DNA and peptide-based vaccines targeting HER2/neu havebeen reported. Amici et al. (U.S. Pat. No. 6,127,344) disclose a methodfor inducing immunity against HER2/neu by administering an expressionvector comprising the full-length human HER2/neu cDNA functionallylinked to the human cytomegalovirus promoter. Morris et al. (WO2004/041065) disclose a method of vaccination with dendritic cellsmodified by adenoviral vectors expressing a non-signaling HER2/neu gene.Cheever and Disis disclose methods for immunizing humans againstHER2/neu-associated cancers with HER2 peptides (U.S. Pat. No.5,846,538). Additionally, HER2/neu peptide-based vaccines have beenstudied in rodent models (for review, see Disis and Cheever, Adv. CancerRes. 71:343-71 (1997)).

The development and commercialization of many vaccines have beenhindered by difficulties associated with obtaining high expressionlevels of exogenous genes in successfully transformed host organisms.Therefore, despite the identification of the wild-type nucleotidesequences encoding hHER2 protein described above, it would be highlydesirable to develop a readily renewable source of human HER2 proteinthat utilizes hHER2-encoding nucleotide sequences that are optimized forexpression in the intended host cell, said source allowing for thedevelopment of a cancer vaccine which is efficacious and not hindered byself-tolerance.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods to elicit orenhance immunity to the protein products expressed by the human HER2gene, which is associated with numerous adenocarcinomas, includingbreast and ovarian cancers. Specifically, the present invention providespolynucleotides encoding human HER2 protein, or a truncated form ofhuman HER2 protein which comprises the extracellular and transmembranedomains of the HER2 protein (hereinafter hHER2ECDTM), wherein saidpolynucleotides are codon-optimized for high level expression in a humancell. The present invention further provides adenoviral andplasmid-based vectors comprising the synthetic polynucleotides anddiscloses use of said vectors in immunogenic compositions and vaccinesfor the prevention and/or treatment of HER2-associated cancer. Thepolynucleotides described herein are more efficient that wild-type HER2in eliciting a cellular and humoral immune response against human HER2.

The present invention also relates to synthetic nucleic acid molecules(polynucleotides) comprising a sequence of nucleotides that encode humanepidermal growth factor 2 antigen (hereinafter hHER2) as set forth inSEQ ID NO:2, wherein the synthetic nucleic acid molecules arecodon-optimized for high-level expression in a human cell (hereinafterhHER2.opt). The present invention further relates to synthetic nucleicacid molecules (polynucleotides) comprising a sequence of nucleotidesthat encode human HER2ECDTM as set forth in SEQ ID NO:14, wherein thesynthetic nucleic acid molecules are codon-optimized for high-levelexpression in a human cell. The nucleic acid molecules disclosed hereinmay be transfected into a host cell of choice wherein the recombinanthost cell provides a source for substantial levels of an expressedfunctional hHER2 protein (SEQ ID NO:2) or hHER2ECDTM protein (SEQ IDNO:14).

The present invention further relates to a synthetic nucleic acidmolecule which encodes mRNA that expresses a human HER2 protein. Apreferred aspect of this portion of the present invention is disclosedin FIG. 1, which shows a DNA molecule (SEQ ID NO:1) that encodes a hHER2protein (SEQ ID NO:2). The preferred nucleic acid molecule of thepresent invention is codon-optimized for high-level expression in ahuman cell. The sequence of this preferred polynucleotide also containsa mutation abolishing tyrosine kinase activity (AAA2257GCC, K753A).Nucleotide sequences that do not contain this mutation are alsocontemplated by this invention.

The present invention additionally relates to a synthetic nucleic acidmolecule which encodes mRNA that expresses a human HER2ECDTM protein. Apreferred aspect of this portion of the present invention is disclosedin FIG. 6A, which shows a DNA molecule (SEQ ID NO:9) that encodes ahHER2ECDTM protein (SEQ ID NO:14). The preferred nucleic acid moleculeof the present invention is codon-optimized for high-level expression ina human cell.

The present invention also relates to recombinant vectors andrecombinant host cells, both prokaryotic and eukaryotic, which containthe nucleic acid molecules disclosed throughout this specification.

The present invention further relates to a process for expressing acodon-optimized human HER2 protein in a recombinant host cell,comprising: (a) introducing a vector comprising a syntheticpolynucleotide encoding a human HER2 protein into a suitable host cell,wherein the synthetic polynucleotide is codon-optimized for optimalexpression in a human cell; and, (b) culturing the host cell underconditions which allow expression of said human HER2 protein.

The present invention also relates to a process for expressing acodon-optimized human HER2ECDTM protein in a recombinant host cell,comprising: (a) introducing a vector comprising a syntheticpolynucleotide encoding a human HER2ECDTM protein into a suitable hostcell, wherein the synthetic polynucleotide is codon-optimized foroptimal expression in a human cell; and, (b) culturing the host cellunder conditions which allow expression of said human HER2ECDTM protein.

Another aspect of this invention is a method of preventing or treatingcancer comprising administering to a mammal a vaccine vector comprisinga synthetic nucleic acid molecule, the synthetic nucleic acid moleculecomprising a sequence of nucleotides that encodes a human epidermalgrowth factor 2 antigen (hHER2) protein as set forth in SEQ ID NO:2, ora human HER2ECDTM protein as set forth in SEQ ID NO:14, wherein thesynthetic nucleic acid molecule is codon-optimized for high levelexpression in a human cell.

The present invention further relates to an adenovirus vaccine vectorcomprising an adenoviral genome with a deletion in the E1 region, and aninsert in the E1 region, wherein the insert comprises an expressioncassette comprising: (a) a codon-optimized polynucleotide encoding ahuman HER2 protein or a human HER2ECDTM protein; and (b) a promoteroperably linked to the polynucleotide.

The present invention also relates to a vaccine plasmid comprising aplasmid portion and an expression cassette portion, the expressioncassette portion comprising: (a) a synthetic polynucleotide encoding ahuman HER2 protein or a human HER2ECDTM protein, wherein the syntheticpolynucleotide is codon-optimized for optimal expression in a humancell; and (b) a promoter operably linked to the polynucleotide.

Another aspect of the present invention is a method of protecting amammal from cancer or treating a mammal suffering from HER2-associatedcancer comprising: (a) introducing into the mammal a first vectorcomprising: i) a codon-optimized polynucleotide encoding a human HER2protein or a human HER2ECDTM protein; and ii) a promoter operably linkedto the polynucleotide; (b) allowing a predetermined amount of time topass; and (c) introducing into the mammal a second vector comprising: i)a codon-optimized polynucleotide encoding a human HER2 protein or ahuman HER2ECDTM protein; and ii) a promoter operably linked to thepolynucleotide.

As used throughout the specification and in the appended claims, thesingular forms “a,” “an,” and “the” include the plural reference unlessthe context clearly dictates otherwise.

As used throughout the specification and appended claims, the followingdefinitions and abbreviations apply:

The term “promoter” refers to a recognition site on a DNA strand towhich the RNA polymerase binds. The promoter forms an initiation complexwith RNA polymerase to initiate and drive transcriptional activity. Thecomplex can be modified by activating sequences termed “enhancers” orinhibiting sequences termed “silencers”.

The term “cassette” refers to a nucleotide or gene sequence that is tobe expressed from a vector, for example, the nucleotide or gene sequenceencoding the HER2 protein or the HER2ECDTM protein. In general, acassette comprises a gene sequence inserted into a vector which, in someembodiments, provides regulatory sequences for expressing the nucleotideor gene sequence. In other embodiments, the nucleotide or gene sequenceprovides the regulatory sequences for its expression. In furtherembodiments, the vector provides some regulatory sequences and thenucleotide or gene sequence provides other regulatory sequences. Forexample, the vector can provide a promoter for transcribing thenucleotide or gene sequence and the nucleotide or gene sequence providesa transcription termination sequence. The regulatory sequences which canbe provided by the vector include, but are not limited to, enhancers,transcription termination sequences, splice acceptor and donorsequences, introns, ribosome binding sequences, and poly(A) additionsequences. The cassette is similar in concept to a cassette tape; eachcassette has its own sequence. Thus by interchanging the cassette, thevector will express a different sequence. Because of the restrictionsites at the 5′ and 3′ ends, the cassette can be easily inserted,removed or replaced with another cassette.

The term “vector” refers to some means by which DNA fragments can beintroduced into a host organism or host tissue. There are various typesof vectors including plasmid, virus (including adenovirus),bacteriophages and cosmids.

The term “first generation,” as used in reference to adenoviral vectors,describes said adenoviral vectors that are replication-defective. Firstgeneration adenovirus vectors typically have a deleted or inactivated E1gene region, and preferably have a deleted or inactivated E3 generegion.

The designation “pV1J-hHER2.opt” refers to a plasmid construct,disclosed herein, comprising the human CMV immediate-early (IE) promoterwith intron A, a full-length codon-optimized human HER2 gene, bovinegrowth hormone-derived polyadenylation and transcriptional terminationsequences, and a minimal pUC backbone (see EXAMPLE 2).

The designation “pV1J-hHER2ECDTM.opt” refers to a plasmid construct,disclosed herein, comprising the human CMV immediate-early (IE) promoterwith intron A, a truncated codon-optimized human HER2 gene comprisingthe extracellular and transmembrane domains of the HER2 gene, bovinegrowth hormone-derived polyadenylation and transcriptional terminationsequences, and a minimal pUC backbone (see EXAMPLE 2).

The designation “pV1J-hHER2.wt” refers to a construct as describedabove, except the construct comprises a wild-type full-length human HER2gene instead of a codon-optimized human HER2 gene.

The designation “pV1J-hER2ECDTM.wt” refers to a construct as describedabove, except the construct comprises a wild-type truncated human HER2gene, said truncated gene comprising a sequence of nucleotides thatencode the extracellular and transmembrane domains of the HER2 protein,instead of a codon-optimized full-length human HER2 gene.

The designations “MRKAd5-hHER2.opt,” “MRKAd5-hHER2ECDTM.opt” and“MRKAd5-hHER2.wt” refer to three constructs, disclosed herein, whichcomprise an Ad5 adenoviral genome deleted of the E1 and E3 regions. Inthe “MRKAd5-hHER2.opt” construct, the E1 region is replaced by acodon-optimized full-length human HER2 gene in an E1 parallelorientation under the control of a human CMV promoter without intron A,followed by a bovine growth hormone polyadenylation signal. The“MRKAd5-hHER2ECDTM.opt” construct is essentially as described above,except the E1 region of the Ad5 genome is replaced by a codon-optimizedtruncated version of the human HER2 gene, said truncated HER2 genecomprising a sequence of nucleotides that encode the extracellular andthe transmembrane domains of the HER2 receptor. The “MRKAd5-hHER2.wt”construct is essentially as described above, except the E1 region of theAd5 genome is replaced with a wild-type fall-length human HER2 sequence(see EXAMPLE 11).

The term “effective amount” means sufficient vaccine composition isintroduced to produce the adequate levels of the polypeptide, so that animmune response results. One skilled in the art recognizes that thislevel may vary.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those prone to havethe disorder or those in which the disorder is to be prevented.

A “disorder” is any condition that would benefit from treatment with themethods or the vaccines and immunogenic compositions described herein.This term includes chronic and acute disorders or diseases includingthose pathological conditions which predispose the mammal to thedisorder in question. The methods and the vaccines of the presentinvention are intended for the treatment of disorders or conditionsassociated with aberrant HER2/neu-associated expression or signaling,including, but in no way limited to, breast, colorectal, gastric,ovarian, and lung cancer.

A “conservative amino acid substitution” refers to the replacement ofone amino acid residue by another, chemically similar, amino acidresidue. Examples of such conservative substitutions are: substitutionof one hydrophobic residue (isoleucine, leucine, valine, or methionine)for another; substitution of one polar residue for another polar residueof the same charge (e.g., arginine for lysine; glutamic acid foraspartic acid).

“hHER2.wt” and “hHER2.opt” refer to a human epidermal growth factor 2antigen and a human codon-optimized epidermal growth factor 2 antigen,respectively.

“hHER2ECDTM.wt” and “hHER2ECDTM.opt” refer to a truncated humanepidermal growth factor 2 antigen and a truncated human codon-optimizedepidermal growth factor 2 antigen, respectively. The truncated forms ofHER2, “hHER2ECDTM.wt” and “hHER2ECDTM.opt,” comprise the extracellularand transmembrane domains of the human HER2 protein.

The term “mammalian” refers to any mammal, including a human being.

The abbreviation “Ag” refers to an antigen.

The abbreviations “Ab” and “mnAb” refer to an antibody and a monoclonalantibody, respectively.

The abbreviation “ORF” refers to the open reading frame of a gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of a codon-optimized polynucleotide(hHER2.opt, SEQ ID NO:1) that encodes human HER2 protein. See EXAMPLE 1.Panel B shows the deduced amino acid sequence of the human HER2 protein(SEQ ID NO:2).

FIG. 2 shows the identification of immunodominant T-cell epitopes in thehuman HER2 protein by ELISPOT and intracellular staining (ICS) analysis.BALB/c mice immunized with Ad5-hHER2 were analyzed for the induction ofhuman HER2-specific cellular immunity. The number of IFN-γ-secretinganti-human HER2 T cells was determined by ELISPOT on splenocytes fromgroups of mice (indicated in the first column) using pools or singlepeptides. Data displayed are representative of several independentexperiments. Values are expressed as the number of spot forming colonies(SFC)/10⁶ total splenocytes, subtracted of the background valuesdetermined in the absence of peptides (typically less than 10 SFC/10⁶total splenocytes). Numbers corresponding to more than three times thebackground measured in control experiments without antigenic peptideswere considered positive values and are indicated in boldface. Frequencyof CD4⁺ or CD8⁺ T-cell secreting IFN-γ was measured by ICS. Datadisplayed are representative of several independent experiments. Valuesare expressed as 1000×[(IFN-γ CD3⁺ and CD4⁺ or CD8⁺)/(CD3⁺ and CD4⁺ orCD8⁺)]. Values higher than 1% were considered positive and indicated inboldface. Sequences encompassed by the pool or by the single peptideused in the assay are indicated on the left. Numbers refers to theposition of amino acid residue of the human HER2 protein.

FIG. 3 shows the in vitro expression of hHER2 following transfection in(A) human embryonic kidney HEK-293 cells and (B) mouse myoblasts C2C7.Data are expressed as the geometric mean of the channel fluorescencefrom which the signal generated by the empty pV1JnsA plasmid has beensubtracted. For C2C7 cells, data are normalized on the efficiency ofpEGFP DNA transfection.

FIG. 4 shows the immune response to human HER2 in BALB/c mice. Panel (A)shows that codon-optimized HER2 yielded significantly improved ELISPOTvalues compared to wild type HER2. Shown are results from immunizationof four groups, each comprising two mice, with plasmid pV1J-hHER2.wt orpV1J-hHER2.opt (50 μg/dose electroinjected in the quadriceps muscle).Two weeks after the last injection, the frequency of IFN-γ secreting Tcells in mouse splenocytes was determined via IFN-γ ELISPOT assay usingpeptides hNeu15.3 (aa 63-71, including a CD8+ epitope), hNeu301 (aa1202-1214, including a CD8+ epitope) and hNeu42 (aa 165-179, including aCD4+ epitope). Results from 2.5×10⁵ and 5×10⁵ splenocytes, with tworeplicas of each amount tested, are shown. Average values werecalculated by subtracting the background level determined in the absenceof peptides (typically less than 10 SFC/10⁶ total splenocytes). Resultswere expressed as the number of SFC/10⁶ total splenocytes. Panel (B)shows that pV1J-hHER2.opt elicits a significantly improved IgG1 andIgG2a humoral response compared to pV1J-hHER2.wt. Serum samples werecollected at week 6 (the day before the first immunization, pre-bleed)and week 14 (two weeks after the last injection) from groups of 4 miceimmunized with pV1J-hHER2.wt or pV1J-hHER2.opt plasmid DNA. Anti-hHER2antibody titers in the pooled sera from each group of mice were measuredby BLISA using the dimeric extra-cellular domain of hHER2 (HER2-ECD) astarget antigen. AP-conjugated goat anti-mouse IgG1 or IgG2a was used todetect bound mouse antibodies.

FIG. 5 shows a comparison of p185-specific T-cell response elicited inmice by immunization with pV1J-HER2 and Ad5-HER2. Wild-type BALB/c miceand BALB/c transgenic mice overexpressing rat HER2 (indicated as NeuT,see Lucchini et al., Cancer Lett 64(3): 203-9 (1992)) were immunized at6 and 9 weeks of age, either with pV1J-hHER2.wt DNA (50 μg/dose,injected in the quadriceps muscle), followed by electrical stimulationor with Ad5-hHER2.wt At 12 weeks of age, the number of IFN-γ-secretinganti-human cells was determined by ELISPOT analysis from pools of miceusing the peptides indicated. Data displayed are representative ofseveral independent experiments. Values are expressed as in FIG. 1.

FIG. 6, panel A, shows the nucleotide sequence of a codon-optimizedpolynucleotide HER2ECDTM.opt, SEQ ID NO:9) that encodes a truncatedhuman HER2 protein, said protein comprising the extracellular andtransmembrane domains of the HER2 protein. Panel B shows a secondpolynucleotide that encodes the extracellular and transmembrane domainsof the HER2 protein, the second polynucleotide comprising “wild-type”nucleotide sequences, which have not been codon optimized(hHER2ECDTM.wt, SEQ ID NO:10).

FIG. 7 shows an analysis of the cell-mediated response induced in rhesusmonkeys immunized with a mixture of three plasmids expressing humanantigens HER2, CEA and EpCAM, said plasmids comprising nucleotidesequences that are codon-optimized for high-level expression in humancells. The same animals were then immunized with a mixture of three Ad5vectors expressing the wild-type sequence of each of the three antigens.Cell-mediated immune response directed against the highly homologous(98.2% sequence similarity) rhesus monkey HER2 protein was measured byIFN-γ ELISPOT every month for one year. Values are expressed as SFC/10⁶PBMC, subtracted of the background values determined in the absence ofpeptides. Values which are significantly different (p<0.05) frombackground, as measured in control experiments without antigenicpeptides, and higher than the arbitrarily chosen threshold of 55 SFC/106PBMC are indicated in boldface.

FIG. 8 shows a comparison of the cell-mediated immune response elicitedin mice by immunization with pV1J-hHER2opt and pV1J-hHER2ECDTM.opt.Values refer to the frequency of IFNγ-secreting spleen cells as measuredby ELISPOT. Data displayed are derived from three animals and arerepresentative of several independent experiments. Values are expressedas SFC/10⁶ total spleen cells, subtracted of the background valuesdetermined in the absence of peptides (typically less than 5 SFC/10⁶spleen cells). Values which are significantly different (p<0.05) frombackground, as measured in control experiments without antigenicpeptides, and are higher than the arbitrarily chosen threshold of 25SFC/10⁶ spleen cells are indicated in boldface.

DETAILED DESCRIPTION OF THE INVENTION

Human epidermal growth factor 2 (hHER2) is commonly associated with anumber of different types of tumors, including breast, ovarian, gastric,and colon carcinomas. The present invention relates to compositions andmethods to elicit or enhance immunity to the protein product expressedby the hHER2 gene, wherein aberrant hHER2 expression is associated withthe carcinoma or its development. Association of aberrant hHER2expression with a carcinoma does not require that the hHER2 protein beexpressed in tumor tissue at all time points of its development, asabnormal hHER2 expression may be present at tumor initiation and not bedetectable late into tumor progression or vice-versa.

To this end, synthetic DNA molecules encoding a full-length human HER2protein or a truncated human HER2 protein, referred to herein asHER2ECDTM, are provided. Said truncated HER2 comprises the extracellularand transmembrane domains of the human HER2 protein. The codons of thesynthetic DNA molecules are designed so as to use the codons preferredby the projected host cell, which in preferred embodiments is a humancell. The synthetic molecules may be used for the development ofplasmid-based vaccines or recombinant adenovirus, which provideeffective immunoprophylaxis against HER2-associated cancer throughneutralizing antibody and cell-mediated immunity. The syntheticmolecules may be used as an immunogenic composition. This inventionprovides polynucleotides which, when directly introduced into avertebrate in vivo, including mammals such as primates and humans,induce the expression of encoded proteins within the animal.

The wild-type human HER2 nucleotide sequence has been reported (Coussenset al., Science 230: 1132-39 (1985); King et al., Science 229: 974-76(1985)). The present invention provides synthetic DNA molecules encodingthe full-length human HER2 protein, or a truncated human HER2 protein,HER2ECDTM, comprising the extracellular and transmembrane domains ofhHER2. The synthetic molecules of the present invention comprise asequence of nucleotides, wherein at least one of the nucleotides hasbeen altered so as to use the codons preferred by a human cell, thusallowing for high-level expression of hHER2 or hHER2ECDTM in a humanhost cell. The synthetic molecules may be used as a source of hHER2 orhHER2ECDTM protein, which may be used in a cancer vaccine to provideeffective immunoprophylaxis against hHER2-associated carcinomas throughneutralizing antibody and cell-mediated immunity. Alternatively, thesynthetic molecules may be used as the basis of a DNA vaccine oradenovirus vaccine.

A “triplet” codon of four possible nucleotide bases can exist in over 60variant forms. Because these codons provide the message for only 20different amino acids (as well as transcription initiation andtermination), some amino acids can be coded for by more than one codon—aphenomenon known as codon redundancy. For reasons not completelyunderstood, alternative codons are not uniformly present in theendogenous DNA of differing types of cells. Indeed, there appears toexist a variable natural hierarchy or “preference” for certain codons inspecific types of cells. As one example, the amino acid leucine isspecified by any of six DNA codons including CTA, CTC, CTG, CTT, TTA,and TTG. Exhaustive analysis of genome codon frequencies formicroorganisms has revealed endogenous DNA of E. coli most commonlycontains the CTG leucine-specifying codon, while the DNA of yeasts andslime molds most commonly includes a TTA leucine-specifying codon. Inview of this hierarchy, it is generally believed that the likelihood ofobtaining high levels of expression of a leucine-rich polypeptide by anE. coli host will depend to some extent on the frequency of codon use.For example, it is likely that a gene rich in TrA codons will be poorlyexpressed in E. coli, whereas a CTG rich gene will probably be highlyexpressed in this host. Similarly, a preferred codon for expression of aleucine-rich polypeptide in yeast host cells would be TTA.

The implications of codon preference phenomena on recombinant DNAtechniques are manifest, and the phenomenon may serve to explain manyprior failures to achieve high expression levels of exogenous genes insuccessfully transformed host organisms—a less “preferred” codon may berepeatedly present in the inserted gene and the host cell machinery forexpression may not operate as efficiently. This phenomenon suggests thatsynthetic genes which have been designed to include a projected hostcell's preferred codons provide an optimal form of foreign geneticmaterial for practice of recombinant DNA techniques. Thus, one aspect ofthis invention is a human HER2 gene that is codon-optimized forhigh-level expression in a human cell. In a preferred embodiment of thisinvention, it has been found that the use of alternative codons encodingthe same protein sequence may remove the constraints on expression ofexogenous hHER2 protein in human cells. Another aspect of this inventionis a truncated human HER2 gene, hHER2ECDTM, that is codon optimized forhigh-level expression in a human host cell, said truncated HER2 genecomprising nucleotide sequences that encode the extracellular andtransmembrane domains of human HER2.

In accordance with this invention, the human HER2 gene sequence and thehuman HER2ECDTM gene sequence were converted to polynucleotide sequenceshaving identical translated sequences as compared to wild-typeequivalents, but with alternative codon usage as described by Lathe,“Synthetic Oligonucleotide Probes Deduced from Amino Acid Sequence Data:Theoretical and Practical Considerations” J. Molec. Biol. 183:1-12(1985), which is hereby incorporated by reference. The methodologygenerally consists of identifying codons in the wild-type sequence thatare not commonly associated with highly expressed human genes andreplacing them with optimal codons for high expression in human cells.Said optimal codons are referred to herein as “human-preferred” codons.The new gene sequence is then inspected for undesired sequencesgenerated by codon replacements (e.g., “ATTTA” sequences, inadvertentcreation of intron splice recognition sites, unwanted restriction enzymesites, high GC content, etc.). Undesirable sequences are eliminated bysubstitution of the existing codons with different codons coding for thesame amino acid. The synthetic gene segments are then tested forimproved expression.

The methods described above were used to create synthetic gene sequencesfor human HER2 and human HER2ECDTM, resulting in full-length andtruncated genes comprising codons optimized for high level expression inhuman cells. While the above procedure provides a summary of ourmethodology for designing codon-optimized genes for use in cancervaccines, it is understood by one skilled in the art that similarvaccine efficacy or increased expression of genes may be achieved byminor variations in the procedure or by minor variations in thesequence. One of skill in the art will also recognize that additionalDNA molecules may be constructed that provide for high levels of hHER2or hHER2ECDTM expression in human cells, wherein only a portion of thecodons of the DNA molecules are codon-optimized. The nucleic acidmolecules of the present invention are substantially free from othernucleic acids.

Accordingly, the present invention relates to a synthetic polynucleotidecomprising a sequence of nucleotides encoding a human HER2 protein, forexample, the human HER2 protein set forth in SEQ ID NO:2, or abiologically active fragment or mutant form of a human HER2 protein, thepolynucleotide sequence comprising codons optimized for expression in ahuman host. Said mutant forms of the hHER2 protein include, but are notlimited to: conservative amino acid substitutions, amino-terminaltruncations, carboxy-terminal truncations, deletions, or additions. Anysuch biologically active fragment and/or mutant will encode either aprotein or protein fragment which at least substantially mimics theimmunological properties of the hHER2 protein as set forth in SEQ IDNO:2. The synthetic polynucleotides of the present invention encode mRNAmolecules that express a functional human HER2 protein so as to beuseful in the development of a therapeutic or prophylactic cancervaccine.

A preferred polynucleotide of the present invention is a polynucleotidecomprising a sequence of nucleotides encoding a truncated humanHER2ECDTM protein (SEQ ID NO:14), the polynucleotide sequence comprisingcodons optimized for expression in a human host. A particularlypreferred polynucleotide of the present invention comprises a sequenceof nucleotides as set forth in SEQ ID NO:9.

The present invention also relates to a synthetic nucleic acid molecule(polynucleotide) comprising a sequence of nucleotides which encodes mRNAthat expresses a human HER2 protein, for example, the full-length humanHER2 protein as set forth in SEQ ID NO:2, or a truncated HER2ECDTMprotein, for example the HER2ECDTM sequence as set forth in SEQ IDNO:14. The synthetic nucleic acid molecules of the present invention arecodon-optimized for high-level expression in a human host cell.

Also included within the scope of the present invention arecodon-optimized polynucleotides comprising a sequence of nucleotidesthat encode a variant HER2 polypeptide that has at least 90% identity tothe amino acid sequence of SEQ ID NO:2, which may include up to Na aminoacid alterations over the entire length of SEQ ID NO:2, wherein Na isthe maximum number of amino acid alterations, and is calculated by theformulaN _(a) =X _(a)−(X _(a) Y),in which X_(a) is the total number of amino acids in SEQ ID NO:2, and Yhas a value of 0.90, wherein any non-integer product of X_(a) and Y isrounded to the nearest integer prior to subtracting such product fromX_(a). Likewise, the present invention also contemplates codon-optimizednucleotide sequences encoding variants of the HER2ECDTM polypeptide asset forth in SEQ ID NO:14.

The present invention also relates to recombinant vectors andrecombinant host cells, both prokaryotic and eukaryotic, which containthe nucleic acid molecules disclosed throughout this specification. Thesynthetic DNA molecules, associated vectors, and hosts of the presentinvention are useful for the development of a cancer vaccine.

A preferred DNA molecule of the present invention comprises thenucleotide sequence disclosed herein as SEQ ID NO:1 (shown in FIG. 1),which encodes the human HER2 protein shown in FIG. 2 and set forth asSEQ ID NO:2. The nucleotide sequence set forth in SEQ ID NO:1 wascodon-optimized for optimal expression in human cells. To avoid PCRamplification problems, in this embodiment of the present invention, aless stringent optimization design was adopted for the hHER2 sequencebetween position 3601 and 3805, which reduced GC content whilepreserving the same amino acid composition. See EXAMPLE 5.

An additional preferred DNA molecule of the present invention comprisesthe nucleotide sequence disclosed herein as SEQ ID NO:9 (shown in FIG.6A), which encodes the human HER2ECDTM protein set forth as SEQ IDNO:14. The nucleotide sequence set forth in SEQ ID NO:9 wascodon-optimized for optimal expression in human cells.

One of skill in the art will realize that other HER2 sequences may bedesigned which are codon-optimized for high-level expression in a humancell, provided that one or more codons are altered to human-preferredcodons. It is preferred that at least about 80% of the codons comprisingthe synthetic HER2 nucleotide sequences of the present invention arehuman-preferred codons. It is more preferred that at least about 85% ofthe codons are human-preferred and even more preferred that at leastabout 90% of the codons are human-preferred.

The present invention also includes biologically active fragments ormutants of SEQ ID NO:1, which encode mRNA expressing human HER2proteins. Any such biologically active fragment and/or mutant willencode either a protein or protein fragment which at least substantiallymimics the pharmacological properties of the hHER2 protein, includingbut not limited to the hHER2 protein as set forth in SEQ ID NO:2. Anysuch polynucleotide includes but is not necessarily limited to:nucleotide substitutions, deletions, additions, amino-terminaltruncations and carboxy-terminal truncations. The mutations of thepresent invention encode mRNA molecules that express a functional hHER2protein in a eukaryotic cell so as to be useful in cancer vaccinedevelopment.

This invention also relates to synthetic codon-optimized DNA moleculesthat encode the hHER2 protein or the hHER2ECDTM protein, wherein thenucleotide sequence of the synthetic DNA differs significantly from thenucleotide sequence of SEQ ID NO:1 or SEQ ID NO:9, but still encodes thehHER2 protein as set forth in SEQ ID NO:2 or the hHER2ECDTM protein asset forth in SEQ ID NO:14. Such synthetic DNAs are intended to be withinthe scope of the present invention. Therefore, the present inventiondiscloses codon redundancy that may result in numerous DNA moleculesexpressing an identical protein. Also included within the scope of thisinvention are mutations in the DNA sequence that do not substantiallyalter the ultimate physical properties of the expressed protein. Forexample, substitution of valine for leucine, arginine for lysine, orasparagine for glutamine may not cause a change in the functionality ofthe polypeptide.

It is known that DNA sequences coding for a peptide may be altered so asto code for a peptide that has properties that are different than thoseof the naturally occurring peptide. Methods of altering the DNAsequences include but are not limited to site directed mutagenesis.Examples of altered properties include but are not limited to changes inthe affinity of an enzyme for a substrate or receptor for a ligand.

The present invention also relates to hHER2opt and hHER2ECDTMopt fusionconstructs, including but not limited to fusion constructs which expressa portion of the human HER2 protein linked to various markers, includingbut in no way limited to GFP (Green fluorescent protein), the MYCepitope, GST, and Fc. Any such fusion construct may be expressed in thecell line of interest and used to screen for modulators of the humanHER2 protein disclosed herein. Also contemplated are fusion constructsthat are constructed to enhance the immune response to human HER2including, but not limited to: DOM, hsp70, and LTB.

The present invention further relates to recombinant vectors thatcomprise the synthetic nucleic acid molecules disclosed throughout thisspecification. These vectors may be comprised of DNA or RNA. For mostcloning purposes, DNA vectors are preferred. Typical vectors includeplasmids, modified viruses, baculovirus, bacteriophage, cosmids, yeastartificial chromosomes, and other forms of episomal or integrated DNAthat can encode a hHER2 protein or a hHER2ECDTM protein. It is wellwithin the purview of the skilled artisan to determine an appropriatevector for a particular gene transfer or other use.

An expression vector containing codon-optimized DNA encoding a hHER2protein may be used for high-level expression of hHER2 in a recombinanthost cell. Additionally, an expression vector containing codon-optimizedDNA encoding a hHER2ECDTM protein may be used for high-level expressionof hHER2ECDTM in a recombinant host cell. Expression vectors mayinclude, but are not limited to, cloning vectors, modified cloningvectors, specifically designed plasmids or viruses. Also, a variety ofbacterial expression vectors may be used to express recombinant hHER2 orhHER2ECDTM in bacterial cells if desired. In addition, a variety offungal cell expression vectors may be used to express recombinant hHER2or hHER2ECDTM in fungal cells. Further, a variety of insect cellexpression vectors may be used to express recombinant protein in insectcells.

The present invention also relates to host cells transformed ortransfected with vectors comprising the nucleic acid molecules of thepresent invention. Recombinant host cells may be prokaryotic oreukaryotic, including but not limited to, bacteria such as E. coli,fungal cells such as yeast, mammalian cells including, but not limitedto, cell lines of bovine, porcine, monkey and rodent origin; and insectcells including but not limited to Drosophila and silkworm derived celllines. Such recombinant host cells can be cultured under suitableconditions to produce hHER2, hHER2ECDTM, or a biologically equivalentform. In a preferred embodiment of the present invention, the host cellis human. As defined herein, the term “host cell” is not intended toinclude a host cell in the body of a transgenic human being, transgenichuman fetus, or transgenic human embryos.

As noted above, an expression vector containing DNA encoding a hHER2protein or a hHER2ECDTM protein may be used for expression of hHER2 orhHER2ECDTM in a recombinant host cell. Therefore, another aspect of thisinvention is a process for expressing a human HER2 protein or a humanHER2ECDTM protein in a recombinant host cell, comprising: (a)introducing a vector comprising a codon-optimized nucleic acid thatencodes human HER2 or human HER2ECDTM into a suitable human host cell;and, (b) culturing the host cell under conditions which allow expressionof said human HER2 protein or said human HER2ECDTM protein.

A preferred embodiment of this aspect of this invention provides aprocess for expressing a human HER2 protein in a recombinant host cell,comprising: (a) introducing a vector comprising a nucleic acid as setforth in SEQ ID NO:1 into a suitable human host cell; and, (b) culturingthe host cell under conditions which allow expression of said human HER2protein.

Another preferred embodiment of this aspect of this invention provides aprocess for expressing a human HER2ECDTM protein in a recombinant hostcell, comprising: (a) introducing a vector comprising a nucleic acid asset forth in SEQ ID NO:9 into a suitable human host cell; and, (b)culturing the host cell under conditions which allow expression of saidhuman HER2ECDTM protein.

Following expression of hHER2 or hHER2ECDTM in a host cell, hHER2 orhHER2ECDTM protein may be recovered to protein in active form. Severalprotein purification procedures are available and suitable for use.Recombinant hHER2 protein or HER2ECDTM protein may be purified from celllysates and extracts by various combinations of, or individualapplication of salt fractionation, ion exchange chromatography, sizeexclusion chromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography. In addition, recombinant proteincan be separated from other cellular proteins by use of animmunoaffinity column made with monoclonal or polyclonal antibodiesspecific for full-length hHER2 protein, or polypeptide fragments ofhHER2 protein.

The nucleic acids of the present invention may be assembled into anexpression cassette which comprises sequences designed to provide forefficient expression of the protein in a human cell. In one embodimentof the invention, the cassette contains a full-length codon-optimizedhHER2 gene, with related transcriptional and translations controlsequences operatively linked to it, such as a promoter, and terminationsequences. In a second embodiment of the invention, the cassettecontains a truncated HER2 gene, H3R2ECDTM, which encodes theextracellular and transmembrane domains of the human HER2 protein. Inpreferred embodiments, the promoter is the cytomegalovirus promoterwithout the intron A sequence (CMV), although those skilled in the artwill recognize that any of a number of other known promoters such as astrong immunoglobulin, or other eukaryotic gene promoter may be used. Apreferred transcriptional terminator is the bovine growth hormoneterminator, although other known transcriptional terminators may also beused. The combination of CMV-BGH terminator is particularly preferred.

In accordance with this invention, the hHER2opt or hHER2ECDTMoptexpression cassette is inserted into a vector. The vector is preferablyan adenoviral vector, although linear DNA linked to a promoter, or othervectors, such as adeno-associated virus or a modified vaccinia virus,retroviral or lentiviral vector may also be used.

If the vector chosen is an adenovirus, it is preferred that the vectorbe a so-called first-generation adenoviral vector. These adenoviralvectors are characterized by having a non-functional E1 gene region, andpreferably a deleted adenoviral E1 gene region. In some embodiments, theexpression cassette is inserted in the position where the adenoviral E1gene is normally located. In addition, these vectors optionally have anon-functional or deleted E3 region. It is preferred that the adenovirusgenome used be deleted of both the E1 and E3 regions (ΔE1E3). Theadenoviruses can be multiplied in known cell lines which express theviral E1 gene, such as 293 cells, or PERC.6 cells, or in cell linesderived from 293 or PERC.6 cell which are transiently or stabilelytransformed to express an extra protein. For examples, when usingconstructs that have a controlled gene expression, such as atetracycline regulatable promoter system, the cell line may expresscomponents involved in the regulatory system. One example of such a cellline is T-Rex-293; others are known in the art.

For convenience in manipulating the adenoviral vector, the adenovirusmay be in a shuttle plasmid form. This invention is also directed to ashuttle plasmid vector which comprises a plasmid portion and anadenovirus portion, the adenovirus portion comprising an adenoviralgenome which has a deleted E1 and optional E3 deletion, and has aninserted expression cassette comprising codon-optimized human HER2 orcodon-optimized hHER2ECDTM. In preferred embodiments, there arerestriction sites flanking the adenoviral portion of the plasmid so thatthe adenoviral vector can easily be removed. The shuttle plasmid may bereplicated in prokaryotic cells or eukaryotic cells.

In a preferred embodiment of the invention, the expression cassette isinserted into the pMRKAd5-HV0 adenovirus plasmid (See Emini et al., WO02/22080, which is hereby incorporated by reference). This plasmidcomprises an Ad5 adenoviral genome deleted of the E1 and E3 regions. Thedesign of the pMRKAd5-HV0 plasmid was improved over prior adenovectorsby extending the 5′ cis-acting packaging region further into the E1 geneto incorporate elements found to be important in optimizing viralpackaging, resulting in enhanced virus amplification. Advantageously,this enhanced adenoviral vector is capable of maintaining geneticstability following high passage propagation.

Standard techniques of molecular biology for preparing and purifying DNAconstructs enable the preparation of the adenoviruses, shuttle plasmids,and DNA immunogens of this invention.

It has been determined in accordance with the present invention that thesynthetic cDNA molecules described herein (e.g. SEQ ID NO:1 and SEQ IDNO:9), which are codon-optimized for high-level expression in a humancell, are expressed with greater efficiency than the corresponding wildtype sequence. Additionally, it was shown herein that hHER2opt is moreimmunogenic that hHER2 and is more efficient in eliciting both cellularand humoral immune responses.

Therefore, the vectors described above may be used in immunogeniccompositions and vaccines for preventing the development ofadenocarcinomas associated with aberrant HER2 expression and/or fortreatment of existing cancers. The vectors of the present inventionallow for vaccine development and commercialization by eliminatingdifficulties with obtaining high expression levels of exogenous HER2 insuccessfully transformed host organisms. To this end, one aspect of theinstant invention is a method of preventing or treating HER2-associatedcancer comprising administering to a mammal a vaccine vector comprisinga synthetic codon-optimized nucleic acid molecule, the syntheticcodon-optimized nucleic acid molecule comprising a sequence ofnucleotides that encodes a human HER2 protein as set forth in SEQ IDNO:2 or a human HER2ECDTM protein as set forth in SEQ ID NO:14.

In accordance with the method described above, the vaccine vector may beadministered for the treatment or prevention of cancer in any mammal. Ina preferred embodiment of the invention, the mammal is a human.

Further, one of skill in the art may choose any type of vector for usein the treatment and prevention method described. Preferably, the vectoris an adenovirus vector or a plasmid vector. In a preferred embodimentof the invention, the vector is an adenoviral vector comprising anadenoviral genome with a deletion in the adenovirus E1 region, and aninsert in the adenovirus E1 region, wherein the insert comprises anexpression cassette comprising: (a) a synthetic codon-optimizedpolynucleotide encoding a human HER2 protein or a human HER2ECDTMprotein; and (b) a promoter operably linked to the polynucleotide.

The instant invention further relates to an adenovirus vaccine vectorcomprising an adenoviral genome with a deletion in the E1 region, and aninsert in the E1 region, wherein the insert comprises an expressioncassette comprising: (a) a synthetic codon-optimized polynucleotideencoding a human HER2 protein or a human HER2ECDTM protein; and (b) apromoter operably linked to the polynucleotide.

In a preferred embodiment of this aspect of the invention, theadenovirus vector is an Ad vector.

In other preferred embodiments of the invention, the vector is an Ad6vector or an Ad24 vector.

In another aspect, the invention relates to a vaccine plasmid comprisinga plasmid portion and an expression cassette portion, the expressioncassette portion comprising: (a) a synthetic codon-optimizedpolynucleotide encoding a human HER2 protein; and (b) a promoteroperably linked to the polynucleotide.

The invention also relates to a vaccine plasmid comprising a plasmidportion and an expression cassette portion, the expression cassetteportion comprising: (a) a synthetic codon-optimized polynucleotideencoding a human HER2ECDTM protein; and (b) a promoter operably linkedto the polynucleotide.

In some embodiments of this invention, the recombinant adenovirusvaccines disclosed herein are used in various prime/boost combinationswith a plasmid-based polynucleotide vaccine in order to induce anenhanced immune response. In this case, the two vectors are administeredin a “prime and boost” regimen. For example the first type of vector isadministered, then after a predetermined amount of time, for example, 2weeks, 1 month, 2 months, six months, or other appropriate interval, asecond type of vector is administered. Preferably the vectors carryexpression cassettes encoding the same polynucleotide or combination ofpolynucleotides. In the embodiment where a plasmid DNA is also used, itis preferred that the vector contain one or more promoters recognized bymammalian or insect cells. In a preferred embodiment, the plasmid wouldcontain a strong promoter such as, but not limited to, the CMV promoter.The synthetic human HER2 gene, HER2ECDTM gene, or other gene to beexpressed would be linked to such a promoter. An example of such aplasmid would be the mammalian expression plasmid V1Jns as described (J.Shiver et. al. in DNA Vaccines, M. Liu et al. eds., N.Y. Acad. Sci.,N.Y., 772:198-208 (1996), which is herein incorporated by reference).

As stated above, an adenoviral vector vaccine and a plasmid vaccine maybe administered to a vertebrate as part of a single therapeutic regimeto induce an immune response. To this end, the present invention relatesto a method of protecting a mammal from cancer comprising: (a)introducing into the mammal a first vector comprising: i) a syntheticcodon-optimized polynucleotide encoding a human HER2 protein or a humanHER2ECDTM; and ii) a promoter operably linked to the polynucleotide; (b)allowing a predetermined amount of time to pass; and (c) introducinginto the mammal a second vector comprising: i) a syntheticcodon-optimized polynucleotide encoding a human HER2 protein or a humanHER2ECDTM; and ii) a promoter operably linked to the polynucleotide.

In one embodiment of the method of protection described above, the firstvector is a plasmid and the second vector is an adenovirus vector. In analternative embodiment, the first vector is an adenovirus vector and thesecond vector is a plasmid.

The instant invention further relates to a method of treating a mammalsuffering from a HER2-associated cancer comprising: (a) introducing intothe mammal a first vector comprising: i) a synthetic codon-optimizedpolynucleotide encoding a human HER2 protein or a human HER2ECDTMprotein; and ii) a promoter operably linked to the polynucleotide; (b)allowing a predetermined amount of time to pass; and (c) introducinginto the mammal a second vector comprising: i) a syntheticcodon-optimized polynucleotide encoding a human HER2 protein or a humanHER2ECDTM protein; and ii) a promoter operably linked to thepolynucleotide.

In one embodiment of the method of treatment described above, the firstvector is a plasmid and the second vector is an adenovirus vector. In analternative embodiment, the first vector is an adenovirus vector and thesecond vector is a plasmid.

The amount of expressible DNA or transcribed RNA to be introduced into avaccine recipient will depend partially on the strength of the promotersused and on the immunogenicity of the expressed gene product. Ingeneral, an immunologically or prophylactically effective dose of about1 ng to 100 mg, and preferably about 10 μg to 300 μg of a plasmidvaccine vector is administered directly into muscle tissue. An effectivedose for recombinant adenovirus is approximately 10⁶-10¹² particles andpreferably about 10⁷-10¹¹ particles. Subcutaneous injection, intradermalintroduction, impression though the skin, and other modes ofadministration such as intraperitoneal, intravenous, or inhalationdelivery are also contemplated. It is also contemplated that boostervaccinations may be provided. Parenteral administration, such asintravenous, intramuscular, subcutaneous or other means ofadministration with adjuvants such as interleukin 12 protein,concurrently with or subsequent to parenteral introduction of thevaccine of this invention is also advantageous.

The vaccine vectors of this invention may be naked, i.e., unassociatedwith any proteins, adjuvants or other agents which impact on therecipient's immune system. In this case, it is desirable for the vaccinevectors to be in a physiologically acceptable solution, such as, but notlimited to, sterile saline or sterile buffered saline. Alternatively, itmay be advantageous to administer an immunostimulant, such as anadjuvant, cytokine, protein, or other carrier with the vaccines orimmunogenic compositions of the present invention. Therefore, thisinvention includes the use of such immunostimulants in conjunction withthe compositions and methods of the present invention. Animmunostimulant, as used herein, refers to essentially any substancethat enhances or potentiates an immune response (antibody and/orcell-mediated) to an exogenous antigen. Said immunostimulants can beadministered in the form of DNA or protein. Any of a variety ofimmunostimulants may be employed in conjunction with the vaccines andimmunogenic compositions of the present inventions, including, but notlimited to: GM-CSF, IFNα, tetanus toxoid, IL12, B7.1, LFA-3 and ICAM-1.Said immunostimulants are well-known in the art. Agents which assist inthe cellular uptake of DNA, such as, but not limited to calcium ion, mayalso be used. These agents are generally referred to as transfectionfacilitating reagents and pharmaceutically acceptable carriers. Those ofskill in the art will be able to determine the particularimmunostimulant or pharmaceutically acceptable carrier as well as theappropriate time and mode of administration.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing methodologies and materials thatmight be used in connection with the present invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to those precise embodiments, and that various changesand modifications may be effected therein by one skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

The following examples illustrate, but do not limit the invention.

EXAMPLE 1

Human HER2 Optimized Codon Sequence.

The entire hHER2.opt coding sequence was synthesized and assembled byBIONEXUS (Bionexus Inc. Oakland Calif.) and cloned into the pCR-bluntvector (Invitrogen, The Netherlands). The hHER2.opt cDNA was constructedusing oligonucleotides and assembled by PCR. For many experimentsdescribed herein, the hHER2.opt nucleotide sequence used carried anoptimized Kozak sequence at its 5′-end, the complete nucleotide sequenceas set forth in SEQ ID NO:11.

In addition, the ATP binding Lysine residue 753 was substituted withAlanine (K753A) by replacing codon AAA with GCA. This mutation abrogatestyrosine kinase activity of the corresponding protein and eliminates thedownstream signaling events and resulting oncogenic activity of human(Messerle et al. Mol Cell Endocrinol 105(1): 1-10 (1994)) or rat HER2(Ben Levi et al., supra). In addition, the kinase-deficient K756A mutantcan inactivate the signaling activity of a co-expressed oncogenichHER2.wt.

EXAMPLE 2

Plasmid Constructs

pV1J-hHER2.wt: The human HER2 wild type coding sequence was amplified byPCR from plasmid pLTR-2/erb-B2 (kindly provided by P. Di Fiore, EuropeanInstitute of Oncology, Milan, Italy; Di Fiore et al. Science 237 (4811):178-82 (1987)) using primers hNeu.for1 (5′-CCAGTTTAAACATTTAAATGCCGCCACCATGGAGCTGGCGGCCT-3′; (SEQ ID NO:3 coding sequence isunderlined) and hNeu.rev2 (5′-GCCGTCGACTTTACACTGGCACGT CCAGACCCA-3′ (SEQID NO:4) and TaKaRa LA Taq polymerase (TaKaRa Otsu, Shiga, Japan). Theamplification product, which incorporates an optimized translation startsite (Kozak, M., J Mol Biol 196(4): 947-50 (1987); Kozak, M., NucleicAcids Res 15(20): 8125-48 (1987)), was digested with PmeI and SalIrestriction enzymes and cloned into the EcoRV and SalI sites ofmammalian expression plasmid pV1JnsA (see Montgomery et al. DNA CellBiol 12(9): 777-83 (1993)). The plasmid pV1J-hHER2 thus generatedcontained the full-length wild type human HER2 sequence under thetranscriptional control of the human cytomegalovirus immediate-earlypromoter with its intron A sequence. The human wild type HER2 codingsequence was followed by the bovine growth hormone polyadenylationsignal sequence. pV1J-hHER2.opt: A 3793 bp EcoRI-SalI fragment wasexcised from plasmid pCR-hHER2opt and cloned into the correspondingsites of plasmid pV1JnsB (Montgomery et al., supra) generating thepV1J-hHER2.opt plasmid.

pV1J-hHER2ECDTM.opt: A 2168 bp fragment was amplified by PCR usingTaKaRa taq) with primers EEcoRV-for (5′-CCAGATATCGAATTCTAGAGCCGCCACCATGGA-3′ (SEQ ID NO:12)) and SalI-rev(5′-GCTGTCGACTTTATCAGATCAGGATGCCGAACACCACGCCC-3′ (SEQ ID NO:13)) from pV1J-hHER2.opt. The resultingfragment was digested with the EcoRV and SalI restriction enzymes andcloned into the corresponding sites of plasmid pV1JnsB (Montgomery etal., supra) generating the pV1J-hHER2ECDTM.opt plasmid.

EXAMPLE 3

Codon Optimized hHER2 cDNA

A synthetic human HER2 gene (hHER2.opt, FIG. 1) was designed toincorporate human-preferred (humanized) codons for each amino acid(hereinafter aa) residue. During assembly of the gene, PCR amplificationconsistently deleted an 86 bp sequence starting from position 3642, dueto the high GC content of the sequence in this region. To overcome thisproblem, a less stringent optimization design was adopted for the hHER2sequence between position 3601 and 3805, which reduced GC content whilepreserving the same aa composition.

The codon optimized cDNA was modified to maintain 83.9% nucleotideidentity to the original clone. The codon optimized cDNAs were clonedinto the pV1J vectors (Montgomery et al., supra), placing in front aKozak optimized sequence (5′-GCCGCCACC-3′, SEQ ID NO:8) and under thecontrol of the human cytomegalovirus (CMV)/intron A promoter plus thebovine growth hormone (BGH) termination signal. The construct was namedpV1J-hHER2opt (see EXAMPLE 2).

EXAMPLE 4

In Vitro Expression of Plasmid Constructs.

The in vitro expression of the pV1J-hHER2.wt and pV1J.hHER2.optconstructs was assessed by transiently transfecting human embryonickidney HEK-293 or mouse myoblasts C2C7 cell lines and detecting humanHER2 expression by flow cytometry. Supercoiled, endotoxin-free plasmidDNA pV1J-hHER2-wt encoding the human HER2 expression cassette used forimmunization was purified from E. coli DH12S cells (Invitrogen,Groningen, The Netherlands) by Qiagen endo-free plasmid Giga Kit(Qiagen, Hilden, Germany).

Plasmids pV1J-hHER2.wt or pV1J-hHER2.opt were lipofectamine-transfected(Gibco BRL Invitrogen, Groningen, The Netherlands) in HEK-293 cells.Similarly, mouse myoblasts C2C7 cells were transfected with a 1:1 or10:1 mixture of plasmid pHygEGFP (BD Biosciences Clontech, PaloAlto,Calif.) and pV1J-hHER2.wt or pV1J-hHER2.opt.

In vitro transfection of HEK-293 or C2.7 cells showed that the codonoptimized sequence dramatically improves hHER2 expression compared tothe wt sequence (FIGS. 3A and 3B).

EXAMPLE 5

Mice Immunization.

Six-week old inbred female BALB/c mice (H-2d; kindly provided by G.Formi, University of Turin) were kept in standard conditions. Mice weretreated in accordance with European union and institutional guidelines.In particular, mice were fully anesthetized with ketamine (Imalgene 500;Merial Italia, Milano, Italy) at 100 mg/kg of body weight and xylazine(Xilor, BIO 98; S. Lazzaro, Bologna, Italy) at 5.2 mg/kg when necessaryfor procedures.

Fifty micrograms of plasmid DNA were electroinjected in a 50 μl volumein mice quadriceps at 6, 8, and 10 weeks of age, as previously described(Rizzuto et al. Proc. Natl. Acad. Sci. U.S.A. 96(11): 6417-22 (1999)).50 μg of pCMV-bNeu optimized or not were injected without incising theskin into both quadriceps muscles (25 μg in 50 μl of physiologicsolution/injection) and electrostimulation (ES) was performed aspreviously described (Zucchelli et al. J. Virol. 74(24): 11598-607(2000); Rizzuto et al., supra). Briefly, electrical shock consisted of10 trains with 1000 bipolar pulses (130V, 75 mA, 200 μs/phase).

Ad injections were carried out in mice quadriceps in 50 μl volumes. Serawere collected at 7 wks (1 wk before first immunization, pre-bleed), and12 wks (two weeks after the last immunization).

EXAMPLE 6

Mouse IFN-γ ELISPOT Assay.

Mouse splenocytes secreting IFN-γ in an antigen-specific manner weredetected using a standard enzyme-linked immunospot (ELISPOT) assay(Miyahira et al. J Immunol Methods 181(1): 45-54 (1995)). Multiscreen96-well MAIP filtration plates (cat. No. MA1PS4510; Millipore, Bedford,Mass.) were coated with an affinity-purified rat anti-mouse IFN-γantibody ((IgG1, clone R4-6A2, cat No. 18181D, Pharmingen, San Diego,Calif.) diluted sterile PBS. After overnight incubation, plates werewashed with PBST (0.005% Tween in PBS) and incubated with R10 medium for2 hrs at 37° C. to block non-specific binding.

Splenocytes were obtained by removing the spleen from euthanized mice ina sterile manner. Spleen disruption was carried out by grating thedissected spleen on a metal grid. Red blood cells were removed byosmotic lysis by adding 1 ml of 0.1×PBS to the cell pellet and vortexingno more than 15 seconds. One ml of 2×PBS was then added and the volumewas brought to 4 ml with 1×PBS. Cells were pelleted by centrifugation at1200 rpm for 10 min at room temp., and the pellet was resuspended in 1ml R10 medium (RPMI 1640 supplemented with 10% fetal calf serum, 2 mML-glutamine, 50 U of penicillin per ml, 50 μg of streptomycin per ml, 10mM HEPES, 50 μM 2-mercaptoethanol). Viable cells were counted usingTürks staining.

Splenocytes derived from the spleen of two or more immunized mice wereincubated for 15 hrs in the presence of 6 μg/ml of a single or a pool ofpeptides, at a density of 2.5-5 10⁵ cells/well. Concanavalin A (ConA)was used as positive internal control for each mouse at 5 μg/ml. Afterextensive washing with PBST, biotinylated rat anti-mouse IFN-gammaantibody (cat No. 18112D, PharMingen; San Diego, Calif.) was added. Theplates were incubated at 4° C. overnight and then washed with PBST priorto the addition of streptavidin-alkaline phosphatase (Cat No. 13043E,PharMingen; San Diego, Calif.). After incubation for 2 hrs at roomtemperature the plates were extensively washed with PBST and developedby incubating with a one-step nitrobluetetrazolium-5-bromo-4-chloro-3-indolylphosphate substrate (cat. No.34042, Pierce, Rockford, Ill.) for 5 to 15 min for development of spots.Rinsing the plates in water stopped the reaction. DMSO and ConcanavalinA (10 μg/ml) were included as background and positive control for eachsample. Spots were counted by computer-assisted imaging analysis (AIDELR02 coupled with AID ELISPOT 2.6 Software, Strassberg, Germany).

The frequency of positive IFN-γ producing splenocytes per total numberof cells plated per well was calculated as the average value of spotsderived from duplicates at two different cell concentrations subtractedof the average value similarly derived from spots measured in controlwells containing non-pulsed splenocytes. Changes in frequency of IFN-γproducing cells were defined as exceeding a 95% confidence boundcalculated from measurements of controls. Differences with a p value<0.05 were considered significant.

EXAMPLE 7

Identification of Immunodominant T-Cell Epitopes in the Human HER2Protein

Three hundred and twelve 15-amino acid peptides, overlapping by 11 aminoacids, were designed to span the entire human HER2 sequence. Thesepeptides, which also included seven peptides designed to overcomeinsolubility problems, were synthesized by SynPep (Dublin, Calif.). Allpeptides were shown to be >90% pure by HPLC and were used without HPLCpurification. Peptides were reconstituted at 35 mg/ml in DMSO. Thosepeptides that did not immediately dissolve were rocked at 37° C. to aiddissolution. If necessary, 1 to 3 additional volume(s) of DMSO wereadded to fully dissolve those peptides that were still not in solutionafter several hours of rocking. Reconstituted peptides were combined sothat each peptide was equally represented in the mix. The finalconcentration of each peptide in the mix was calculated to be 1 mg/ml.Each mix was aliquoted and stored at 80° C.

To identify the immunodominant T-cell epitopes of the human HER2 gene inBALB/c mice (H-2^(d) genetic background), 6-week-old female BALB/c micewere immunized by injecting 10⁹ vp of Ad5-hHER2 in the quadricepsmuscles. A second injection was performed after 3 weeks. A second groupof mice was similarly injected with saline solution as negative control.Three weeks after the second injection, the animals were sacrificed andthe frequency of IFN-γ secreting T cells in mouse splenocytes wasevaluated by interferon-γ enzyme-linked immunospot (IFN-γ ELISPOT)assay.

Three hundred and eleven peptides, each 15 amino acids long, overlappingby 11 residues, and spanning the entire human HER2 protein sequence werecombined into eleven pools indicated with alphabetical letters from A toK, from N- to C-terminus. Each of these pools was tested for its abilityto stimulate IFN-γ spleen T cells. For peptide pools A, B and M, IFN-γELISPOT measured a statistically significant IFN-γ production by miceimmunized with Ad5-hHER2 as compared to control in the absence ofpeptide. To identify the individual peptide responsible for theactivity, peptides from pool A, B and M were divided into sub-pools,among which A_(III) and A_(IV), B_(III) and M_(I) scored positive.Single peptides from these positive subpools were then tested for theirability to trigger IFN-γ release. Overlapping peptides hNeu-15 andhNeu-16 exhibited high and comparable reactivity. A much lowerreactivity was exhibited by overlapping peptides hNeu-41 and hNeu-42.Another peptide, hNeu-301, was also shown to contain a T-cell epitope.

To confirm these data and identify the CD4+ or CD8+ T cell subsetresponsible for IFN-γ production, IFN-γ secreting T-cells werecharacterized by intracellular staining (ICS). Mouse splenocytes wereincubated with single peptides for 12 hrs in the presence of thesecretion inhibitor brefeldin A, fixed, permeabilized and then stainedfor intracellular IFN-γ, CD3 and CD4 or CD8 markers and analyzed by flowcytometry. ICS confirmed the reactivity of peptide hNeu-15, identifyingit as an epitope able to activate CD8⁺ cells. Peptide hNeu15 and hNeu16were equally reactive in ELISPOT analysis, suggesting that the CD8⁺epitope should be comprised in the 11aa residues common to the twopeptides.

To identify the target nonamer sequence, we tested three 9 aa-longpeptides spanning the overlapping region between hNeu15 and hNeu16.hNeu15.3 proved the most reactive, displaying a slightly increasedreactivity compared to the 15 aa long peptides hNeu-15 and hNeu-16.Interestingly, about half of the reactivity was also detected withhNeu15.1, indicating that two overlapping but distinct CD8⁺ epitopesco-exist in this 11 aa sequence.

IFN-γ ICS analysis also confirmed the reactivity of hNeu301 and typed itas a CD8⁺ epitope. Analysis of these CD8⁺ epitopes by IFN-γ ELISPOTconfirmed the results obtained by ICS. Finally, a low reactivity wasdetected for hNeu41 and hNeu42 peptides, whose low response waspredominantly CD4+.

EXAMPLE 8

Intracellular Cytokine Staining.

Intracellular IFN-γ production was measured according to BD Pharmingenstandard protocol. Briefly, 2×10⁶ spleen cells were cultured for 15 hrsin R10 medium in the presence of 6 μg/ml of single or pool of peptidesand Brefeldin A as protein transport inhibitor (Cytofix/Cytoperm Pluswith GolgiPlug™ Kit; BD Pharmingen; San Diego, Calif.). StaphylococcusEnterotoxin B (SEB) at 10 μg/ml (cat. No. S4881, SIGMA, Saint Louis,Mich.) and DMSO were tested with the splenocytes as positive andbackground control, respectively.

Before staining of surface antigens, Ab anti-mouse CD16/CD32 was used toreduce non-specific immunofluorescent signal (cat No. 553142, BDPharMingen, San Diego, Calif.). The specific signal was obtained withAPC-anti-mouse CD3e, PE-anti-mouse CD4 and PerCP-anti-mouse CD8a (cat.No. 553066, 553653 and 553036, BD Pharmingen; San Diego, Calif.). Thecells were then washed, fixed, permeabilized, and stained forintracellular IFN-γ using FITC-conjugated mAb (cat. No.554411,BDPharmingen San Diego, Calif.). T lymphocyte IFN-γ was calculated as1000×[(IFN-γ⁺, CD3⁺ and CD4⁺ or CD8⁺)/(CD3⁺ and CD4⁺ or CD8⁺)].Generally, at least 50,000 CD3⁺ lymphocytes were collected bysimultaneously gating on CD3⁺ events and small lymphocytes. All sampleswere acquired within 24 hrs of staining using a FACSCalibur flowcytometer and CellQuest software (Becton Dickinson, San Jose, Calif.).

EXAMPLE 9

Antibody Titration and Isotyping.

Sera for antibody titration were obtained by retro-orbital bleeding.ELISA plates (Nunc Maxisorp™, Roskilde, Denmark) were coated overnightat 4° C. with goat anti-human IgG Fc-specific (Pierce; Cat.n. 31123) ata concentration of 2 μg/ml in 50 mM NaHCO₃ (pH 9.6). Excess of antibodywas removed and non-specific binding blocked by incubating for 66 min at37° C. in PBBST5 buffer (BSA 5%. Tween 0.05%). After washing,supernatant of IgB2-cells was added in saturating condition andincubated at RT for 2 hrs (Chen et al. J Biol Chem 271(13): 7620-9(1996)). IgB2 cells (kindly provided by Dr. Y. Yarden, WeizmannInstitute of Science, Rehovot, Israel) are HEK-293 cells secreting thedimeric fusion between the extracellular domain of HER2 and the Fcportion of human Ig. Plates were washed and serial dilution of sera(from 1:4,000 to 1:25,600) in PBBST1 buffer (BSA 1%, Tween 0.05%) wereincubated overnight at 4° C. Pre-immune sera were used as background.Washes were carried out with PBBST1. Secondary antibody (goat anti-mouseIgG1 or IgG2a AP-conjugated (Pharmingen, 557272 and 553389) was diluted1:40,000 in PBBST5 and incubated 2-3 hr at room temp. on a shaker. Afterwashing, plates were developed by incubation with Sigma 106 phosphatasesubstrate (Sigma; cat.n. A106) in diethanolamine. Plates were read by anautomated ELISA reader (Labsistems Multiskan Bichromatic, Helsinki,Finland) and the results were expressed as A=A_(405nm)-A_(620nm). Foreach sample, the background signal detected with the pre-immune serumwas subtracted.

Anti-hHER2 serum titers were calculated as the reciprocal limitingdilution of serum producing an absorbance at least 3-fold greater thanthe absorbance of autologous pre-immune serum at the same dilution.

EXAMPLE 10

Increased Immunogenicity of hHER2opt

To examine in vivo immune responses induced by the wild type and codonoptimized hHER2 expression vectors, BALB/c mice were immunizedintramuscularly with pV1J-hHER2.wt or pV1J-hHER2.opt plasmid DNAfollowed by ES (as described in EXAMPLE 5). Mice were subjected to threeinjections at 6, 8, and 10 weeks of age. Two weeks after the lastimmunization, splenocytes were isolated from each mouse. To quantify theIFNγ-secreting hHER2-specific CD8 T-cell precursor frequencies generatedby the plasmid DNA immunization, the ELISPOT assay for the H-2^(d)restricted T-cell epitope hNeu15.3 and hNeu42 was used. Immunizationwith the HER2 wild-type sequence elicited a barely detectable CD8+response, and reactivity with the CD4+ peptide was absent. In contrast,the optimized HER2 sequence induced a 10-fold enhanced response to theCD8+ peptide, yielding up to 286 IFNγ spot forming cells (SFC, meanvalue) specific for the tested epitopes. A lower CD4+ activity was alsodetected. No peptide-specific IFNγ SFC were detected in thepV1J-nsB-immunized mice (data not shown).

Sera from the same mice were tested by ELISA using the IgB2 protein assubstrate (FIG. 4B). The hHER2-specific antibody titer was detected inall pV1J-hHER2.opt-immunized mice and the geometric mean value of the Abtiter was 46,000 or 78,000 for IgG1 or IG2a. In contrast, thepV1J-hHER2.wt immunized group showed an approximately 100-fold lowergeometric mean titer of hHER2-specific antibody. Thus, these resultsdemonstrate that the codon optimized cDNA of hHER2 is more efficient ineliciting a cellular and humoral immune response than the wild-typesequence.

EXAMPLE 11

Adenovirus Vectors

MRKAd5-hHER2.wt: A SwaI-SalI DNA fragment from pV1J_hHER2 containing thehuman HER2 cDNA was cloned in the corresponding sites of the shuttleplasmid polyMRKΔE1 (Bett et al., Proc Natl Acad Sci USA91(19):8802-04(1994)). The resulting plasmid pMRKΔE1_hHER2 contained ahuman CMV promoter driving the expression of the human HER2 cDNA,followed by the bovine growth hormone polyadenylation signal. PlasmidpMRKΔE1_hHER2 was recombined with the adenoviral backbone plasmidpAd5_HV0 to generate the pre-adenoviral plasmid pAd5-hHER2 wt.

MRKAd5-hHER2ECDTM.opt: Plasmid pCR-hHER2opt was digested with EcoRI. Theresulting 2156 bp insert was purified and cloned into the EcoRI of thepolyMRK-Ad5 shuttle plasmid (See Einini et al., WO 02/22080, which ishereby incorporated by reference).

Plasmids pAd5-hHER2.wt and pMRKAd5-hHER2.opt were linearized by PacIdigestion and transfected into PerC6 cells to generate Ad5-hHER2recombinant adenovirus. The viruses were grown in large quantities bymultiple rounds of amplification and purified by caesium chloridegradient ultra-centrifugation (Fallaux et al., Hum Gene Ther 9(13):1909-17 (1998)). Viral DNA was extracted by proteinase K digestion andgenomic integrity was confirmed by restriction analysis.

HEK 293 cells were infected with MRKAd5-hHER2.wt orMRKAd5-hHER2ECDTM.opt using various multiplicity of infection (m.o.i.).Expression monitored by western blot analysis revealed more than a10-fold difference between the truncated protein expressed from thecodon-optimized sequence compared to the full length protein expressedfrom the wt HER2 sequence (data not shown).

EXAMPLE 12

Comparison of Immunization Regimens

The efficiency in inducing an anti-human HER2 cell-mediated immuneresponse of Adenovirus was compared with that of plasmid DNA associatedwith electrical stimulation, both harboring a CMV-HER2 expressioncassettes. Fifty 1 ug of plasmid pV1J-HER2 were injected into thequadriceps muscle of wt BALB/c mice or BALB/c transgenic miceover-expressing rat HER2 (indicated as NeuT, see Lucchini et al., CancerLett 64(3): 203-9 (1992)) followed by ES at 6 and 9 weeks of age. Twoweeks after boosting, animals were sacrificed and spleen cells werecollected and stimulated with peptides containing the immunodominantp185 epitopes. Very low spot forming cells (SFC) were detected followingstimulation with human peptides, both in BALB/c and neuT mice (FIG. 5).On average, the response was 50-fold lower than that induced by Ads-HER2immunization. The above data show that immunizing mice with plasmidpV1J-HER2 induced a comparable response in BALB/c and neuT mice for eachprotocol.

EXAMPLE 13

Immunization of Rhesus Macaques with Human HER2 in Combination withHuman CEA and EpCAM Antigens

To assess the efficiency of immunization of rhesus macaques (macacamulatta) with the human tumor antigen HER2 in combination with othertumor antigens, a group of 4 rhesus monkeys (2 males and 2 females) wereimmunized with a mixture of plasmid DNA vectors expressing codonoptimized sequences of human tumor antigens Ep-CAM, CEA, and HER2/neu.

Immunization studies were performed at the Biomedical Primate ResearchCentre (BPRC, Rijswijk, The Netherlands). These immunization studieswere designed to evaluate the T cell responses induced by the humanantigens against the rhesus homologues of the same antigens.

Monkeys were vaccinated intramuscularly with injections at weeks 0, 2,4, 6, 8, 10, 12, 14, and 16, followed by electrostimulation. Animalswere injected under anesthesia with 1 ml solution (split over 2 siteswith 0.5 ml/site) containing 6 mg plasmid DNA for animals weighing 2-5kilos.

For electrostimulation, 2 trains of 100 square bipolar pulses (1 seceach), were delivered every other second for a total treatment time of 3sec. The pulse length was 2 msec/phase with a pulse frequency andamplitude of 100 Hz and 100 mA (constant current mode), respectively.

The same monkeys were then vaccinated at weeks 27 and 31 with a mixtureof three Adenovirus 5 (ΔE1-ΔE3, “first generation”, P2 level) expressingthe wt sequence of human HER2, human CEA or human EpCAM, respectively.

To measure the immune response to rhesus homologue of human HER2 usingthe above immunization protocol, blood samples were collected every fourweeks for a total duration of one year. The cell-mediated immuneresponse was measured by IFNγ Elispot assay. Results reported in FIG. 7indicate that the immunization protocol discussed above was effective ininducing a specific immune response against endogenous rhesus homologueof human HER2/neu.

EXAMPLE 14

Comparison of p185-Specific T-Cell Response Elicited in Mice byImmunization with pV1J-HER2opt and pV1J-HER2ECDTM.opt

The immunization efficiency of a C-terminal deletion mutant of p185retaining the extra-cellular and the transmembrane domain (HER2ECDTM)was evaluated. Plasmid DNA expressing the codon-optimized sequence ofthe full length (pV1J-HER2.opt) or of the truncated protein(PV1J-HER2ECDTM.opt) was electro-injected at weeks 10 and 12, andanalysis was performed at week 14. The truncated protein HER2ECDTMinduced an anti-p185 cell-mediated response higher than that induced bythe full length p185 protein, both as CD4+ and CD8+ reactivity, asmeasured by IFN-gamma ELISPOT analysis (FIG. 8). In vitro expressionanalysis in C2.7 murine myoblasts did not reveal differences in theexpression between the two plasmids (data not shown).

1. A synthetic nucleic acid molecule comprising a sequence ofnucleotides that encodes a human HER2/neu protein as set forth in SEQ IDNO:2, the synthetic nucleic acid molecule being codon-optimized for highlevel expression in a human cell.
 2. The synthetic nucleic acid moleculeof claim 1 wherein the sequence of nucleotides comprises the sequence ofnucleotides set forth in SEQ ID NO:1.
 3. A vector comprising the nucleicacid molecule of claim
 1. 4. A host cell comprising the vector of claim3.
 5. A synthetic nucleic acid molecule comprising a sequence ofnucleotides that encodes a variant human HER2/neu polypeptide that hasat least 90% identity to the amino acid sequence of SEQ ID NO:2, whichmay include up to Na amino acid alterations over the entire length ofSEQ ID NO:2, wherein Na is the maximum number of amino acid alterations,and is calculated by the formulaN _(a) =X _(a)−(X _(a) Y), in which X_(a) is the total number of aminoacids in SEQ ID NO:2, and Y has a value of 0.90, wherein any non-integerproduct of X_(a) and Y is rounded to the nearest integer prior tosubtracting such product from X_(a), wherein the sequence of nucleotidesis codon-optimized for high level expression in a human cell.
 6. Asynthetic nucleic acid molecule comprising a sequence of nucleotidesthat encodes a human HER2ECDTM protein as set forth in SEQ ID NO:14, thesynthetic nucleic acid molecule being codon-optimized for high levelexpression in a human cell.
 7. The synthetic nucleic acid molecule ofclaim 6 wherein the sequence of nucleotides comprises the sequence ofnucleotides set forth in SEQ ID NO:9.
 8. A vector comprising the nucleicacid molecule of claim
 6. 9. A host cell comprising the vector of claim8.
 10. A synthetic nucleic acid molecule comprising a sequence ofnucleotides that encodes a variant human HER2ECDTM polypeptide that hasat least 90% identity to the amino acid sequence of SEQ ID NO:14, whichmay include up to N_(a) amino acid alterations over the entire length ofSEQ ID NO:14, wherein N_(a) is the maximum number of amino acidalterations, and is calculated by the formulaN ^(a) =X _(a)−(X _(a) Y), in which X_(a) is the total number of aminoacids in SEQ ID NO:14, and Y has a value of 0.90, wherein anynon-integer product of X_(a) and Y is rounded to the nearest integerprior to subtracting such product from X_(a), wherein the sequence ofnucleotides is codon-optimized for high level expression in a humancell.
 11. A process for expressing a human HER2/neu protein in arecombinant host cell, comprising: (a) introducing a vector comprisingthe nucleic acid of claim 1 into a suitable host cell; and, (b)culturing the host cell under conditions which allow expression of saidhuman HER2 protein.
 12. A process for expressing a human HER2ECDTMprotein in a recombinant host cell, comprising: (a) introducing a vectorcomprising the nucleic acid of claim 6 into a suitable host cell; and,(b) culturing the host cell under conditions which allow expression ofsaid human HER2ECDTM protein.
 13. A method of preventing or treatingHER2-associated cancer comprising administering to a human a vaccinevector comprising a synthetic codon-optimized nucleic acid molecule, thenucleic acid molecule comprising a sequence of nucleotides that encodesa human HER2/neu protein as set forth in SEQ ID NO:2 or a humanHER2ECDTM protein as set forth in SEQ ID NO:14.
 14. (canceled)
 15. Amethod according to claim 13 wherein the vector is an adenovirus vectoror a plasmid vector.
 16. A method according to claim 15 wherein thevector is an adenoviral vector comprising an adenoviral genome with adeletion in the adenovirus E1 region, and an insert in the adenovirus E1region, wherein the insert comprises an expression cassette comprising:(a) a codon-optimized polynucleotide encoding a human HER2 protein or ahuman HER2ECDTM protein; and (b) a promoter operably linked to thepolynucleotide.
 17. A method according to claim 15 wherein the vector isa plasmid vaccine vector, which comprises a plasmid portion and anexpressible cassette comprising: (a) a codon-optimized polynucleotideencoding a human HER2 protein or a human HER2ECDTM protein; and (b) apromoter operably linked to the polynucleotide.
 18. An adenovirusvaccine vector comprising an adenoviral genome with a deletion in the E1region, and an insert in the E1 region, wherein the insert comprises anexpression cassette comprising: (a) a codon-optimized polynucleotideencoding a human HER2 protein or encoding a human HER2ECDTM protein; and(b) a promoter operably linked to the polynucleotide.
 19. An adenovirusvector according to claim 18 which is an Ad 5 vector.
 20. An adenovirusvector according to claim 18 which is an Ad 6 vector or an Ad 24 vector.21. A vaccine plasmid comprising a plasmid portion and an expressioncassette portion, the expression cassette portion comprising: (a) acodon-optimized polynucleotide encoding a human HER2 protein or a humanHER2ECDTM protein; and (b) a promoter operably linked to thepolynucleotide.
 22. A method of treating a mammal suffering fromHER2-associated cancer comprising: (a) introducing into the mammal afirst vector comprising: i) a codon-optimized polynucleotide encoding ahuman HER2 protein or a human HER2ECDTM protein; and ii) a promoteroperably linked to the polynucleotide; (b) allowing a predeterminedamount of time to pass; and (c) introducing into the mammal a secondvector comprising: i) a codon-optimized polynucleotide encoding a humanHER2 protein or a human HER2ECDTM protein; and ii) a promoter operablylinked to the polynucleotide.
 23. A method according to claim 22 whereinthe first vector is a plasmid and the second vector is an adenovirusvector.
 24. A method according to claim 22 wherein the first vector isan adenovirus vector and the second vector is a plasmid.